Galet Colette, Guillou Florian, Foulon-Gauze Florence, Combarnous Yves, Chopineau Maryse
Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, The University of Iowa, 2-319B BSB, 51 Newton Road, Iowa City, Iowa 52242-1109, USA.
J Endocrinol. 2009 Oct;203(1):167-74. doi: 10.1677/JOE-09-0141. Epub 2009 Jul 9.
The dual LH and FSH activity of the equine LH (eLH)/equine chorionic gonadotropin (eCG) in heterologous species makes eLH/CG a good model to study structure/function relationships of gonadotropins. In order to bypass the problem of intracellular association of the heterodimer, a recombinant single-chain beta alpha eLH/CG was used to identify sequences in the beta-subunit involved in the secretion and activities of the hormone. The C-terminal region of the beta-subunit was progressively truncated. All resulting truncated single-chains were secreted in the media as detected by an anti-beta peptide antibody in reducing conditions. However, using a conformation sensitive ELISA we show that the truncated single-chains were differently recognized: deletion of the last 40 amino acids of the beta-subunit (beta109alpha eLH/CG) resulted in a 90% decrease in the recognized correctly folded hormone compared with the full-length beta alpha eLH/CG single-chain and no properly folded hormone was detected in the secretion medium when the last 46 amino acids of the beta-subunit were deleted (beta103alpha eLH/CG). We thus focused on the six amino acids sequence 104-109, which belongs to the seat-belt region. Mutation of the 104-109 sequence in alanines in the full-length beta alpha eLH/CG (beta104-109Ala alpha) led to a 50% decrease in the production of properly folded hormone in COS-7 as well as in alphaT3 pituitary cells. Moreover, the FSH activity of this mutant was decreased by 70% whereas its LH activity remained intact. These data lead us to conclude that the 104-109 region of the beta eLH/CG subunit is essential for the secretion of a fully folded beta alpha eLH/CG and for its FSH activity but not for its LH activity.
马促黄体素（eLH）/马绒毛膜促性腺激素（eCG）在异种动物中具有促黄体素（LH）和促卵泡素（FSH）的双重活性，这使得eLH/CG成为研究促性腺激素结构/功能关系的良好模型。为了绕过异二聚体在细胞内缔合的问题，一种重组单链β-α eLH/CG被用于鉴定β亚基中参与激素分泌和活性的序列。β亚基的C末端区域被逐步截短。在还原条件下，通过抗β肽抗体检测发现，所有产生的截短单链都分泌到了培养基中。然而，使用构象敏感ELISA我们发现，截短的单链被不同程度地识别：与全长β-α eLH/CG单链相比，β亚基最后40个氨基酸缺失（β109α eLH/CG）导致正确折叠激素的识别率降低90%，当β亚基最后46个氨基酸缺失时（β103α eLH/CG），在分泌培养基中未检测到正确折叠的激素。因此，我们将重点放在了属于安全带区域的104-109这六个氨基酸序列上。全长β-α eLH/CG（β104-109Alaα）中104-109序列突变为丙氨酸，导致COS-7细胞以及αT3垂体细胞中正确折叠激素的产生减少50%。此外，该突变体的FSH活性降低了70%，而其LH活性保持不变。这些数据使我们得出结论，β eLH/CG亚基的104-109区域对于完全折叠的β-α eLH/CG的分泌及其FSH活性至关重要，但对其LH活性并非如此。
